This invention relates to a reagent for measurement of leukocytes and hemoglobin concentration in a blood sample.
Measuring the leukocyte count and hemoglobin concentration in the blood is very important for the clinical diagnosis of leukemia, anemia, etc. or for monitoring the clinical course in patients.
Currently, the leukocyte count and hemoglobin concentration can be measured in a short time by an automatic blood analyzer, and such an analyzer is in widespread use.
Automatic blood analyzers can be roughly classified into an optical detection system for detecting light scattering or fluorescence, and an electric resistance detection system for detecting a change in impedance occurring when particles pass through an aperture. The latter system is superior in terms of ease of use. According to the electric resistance detection system, the measurement of the leukocyte count is performed by adding a hemolytic agent to a blood sample to lyse erythrocytes and prepare a sample for leukocyte measurement with only leukocytes left, and then flowing this sample through a detector to detect a signal issued.
Normally, leukocytes in the peripheral blood include five types of cells, lymphocyte, monocyte, neutrophil, eosinophil and basophil. By formulating the hemolytic agent in a preferred manner, it becomes possible to classify the leukocytes into 2 or 3 populations, or detect only leukocytes of a particular type. Thus, leukocyte classification that has necessarily relied on microscopic observation can be performed automatically in a short time. This has reduced the burden on laboratory technicians, and has obviated the need for special technique for laboratory examination.
To measure hemoglobin concentrations, on the other hand, it has been adopted as the international standard method to convert hemoglobin into cyanmethemoglobin by the use of Van Kampen reagent containing a cyanogen compound, and measuring its absorbance at about 541 nm. For an automatic blood cell analyzer, a method is known which fragments erythrocytes, and denatures hemoglobin, with a quaternary ammonium salt having surface activity, obtains a cyanmethemoglobin-like absorption curve with the use of an alkali cyanide, and determines hemoglobin as well as leukocytes. However, harmful cyanogen compounds are contained in the reagents. These reagents or the samples after measurement need to be subsequently made atoxic before being discarded.
There is a known reagent which permits leukocyte counting without using a cyanogen compound for the determination of the hemoglobin concentration (U.S. Pat. No. 4,185,964). This reagent contains a water-soluble quaternary ammonium salt having surface activity, and a small amount of a polycarboxylic acid having up to about 8 carbon atoms in an amount sufficient to inhibit the lysis of leukocytes. Hemolytic agents which enable leukocytes to be classified into three populations are also known. These hemolytic agents contain quaternary ammonium salts and specific hemoglobin stabilizers (Japanese Unexamined Patent Publication Nos. 3-137566, 3-252557 and 4-13969).
The above hemolytic agents can denature hemoglobin rapidly, but have the drawback that the hemoglobin concentration markedly varies with the liquid temperature of the blood sample. Particularly in U.S. Pat. No. 4,185,964, the variations are great, since no stabilizer is used. In Japanese Unexamined Patent Publication Nos. 3-137566, 3-252557 and 4-13969, specific hemoglobin stabilizers are incorporated, so that the variations are reduced. However, when the liquid temperature fluctuates, the hemoglobin concentration also fluctuates. The stabilizing effect of these stabilizers is insufficient in this case. To obtain stable measurements, therefore, a unit for keeping the liquid temperature constant is required. This is one of the factors leading to an increase in the cost of the apparatus.